Wingless gradient formation in the Drosophila wing

BACKGROUND: Secreted signaling proteins of the Wingless (Wg)/Wnt, Hedgehog and bone morphogenetic protein (BMP)/Decapentaplegic (Dpp) families function as morphogens to control growth and pattern formation during development. Although these proteins have been shown to act directly on distant cells in the developing limbs of the fruit fly Drosophila, little is known about how ligand gradients form in vivo. Wg protein is found in vesicles in Wg-responsive cells in the embryo and imaginal discs. It has been proposed that Wg may be transported by a vesicle-mediated mechanism. RESULTS: A novel method to visualize extracellular Wg protein was used to show that Wg forms an unstable gradient on the basolateral surface of the wing imaginal disc epithelium. Wg movement did not require internalization by dynamin-mediated endocytosis. Dynamin activity was, however, required for Wg secretion. By reversibly blocking Wg secretion, we found that Wg moves rapidly to form a long-range extracellular gradient. CONCLUSIONS: The Wg morphogen gradient forms by rapid movement of ligand through the extracellular space, and depends on continuous secretion and rapid turnover. Endocytosis is not required for Wg movement, but contributes to shaping the gradient by removing extracellular Wg. We propose that the extracellular Wg gradient forms by diffusion.     

Wingless promotes cell survival but constrains growth during Drosophila wing development

During animal development, organs grow to a fixed size and shape. Organ development typically begins with a rapid growth phase followed by a gradual decline in growth rate as the organ matures, but the regulation of either stage of growth remains unclear. The Wnt/Wingless (Wg) proteins are critical for patterning most animal organs, have diverse effects on development and have been proposed to promote organ growth. Here we report that contrary to this view, Wg activity actually constrains wing growth during Drosophila melanogaster wing development. In addition, we demonstrate that Wg is required for wing cell survival, particularly during the rapid growth phase of wing development. We propose that the cell-survival- and growth-constraining activities of Wg function to sculpt and delimit final wing size as part of its overall patterning programme.     

Nemo is an inducible antagonist of Wingless signaling during Drosophila wing development

The cellular events that govern patterning during animal development must be precisely regulated. This is achieved by extrinsic factors and through the action of both positive and negative feedback loops. Wnt/Wg signals are crucial across species in many developmental patterning events. We report that Drosophila nemo (nmo) acts as an intracellular feedback inhibitor of Wingless (Wg) and that it is a novel Wg target gene. Nemo antagonizes the activity of the Wg signal, as evidenced by the finding that reduction of nmo rescues the phenotypic defects induced by misexpression of various Wg pathway components. In addition, the activation of Wg-dependent gene expression is suppressed in wing discs ectopically expressing nmo and enhanced cell autonomously in nmo mutant clones. We find that nmo itself is a target of Wg signaling in the imaginal wing disc. nmo expression is induced upon high levels of Wg signaling and can be inhibited by interfering with Wg signaling. Finally, we observe alterations in Arm stabilization upon modulation of Nemo. These observations suggest that the patterning mechanism governed by Wg involves a negative feedback circuit in which Wg induces expression of its own antagonist Nemo.     

Notch and Wingless modulate the response of cells to Hedgehog signalling in the Drosophila wing

During Drosophila wing development, Hedgehog (Hh) signalling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signalling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. Here, we describe a functional hierarchy of these signalling pathways that highlights the importance of competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc revealed that collier/knot (col/kn), patched (ptc), and decapentaplegic (dpp) are repressed at the DV boundary by N signalling. Attenuation of Hh signalling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. We show that this down-regulation depends on activity of the canonical Wg signalling pathway. We propose that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. Our findings suggest a paradigm of repression of the Hh response by N and/or Wnt signalling that may be applicable to signal integration in vertebrate appendages.     

Regulation of Wingless and Vestigial expression in wing and haltere discs of Drosophila

In the third thoracic segment of Drosophila, wing development is suppressed by the homeotic selector gene Ultrabithorax (Ubx) in order to mediate haltere development. Previously, we have shown that Ubx represses dorsoventral (DV) signaling to specify haltere fate. Here we examine the mechanism of Ubx-mediated downregulation of DV signaling. We show that Wingless (Wg) and Vestigial (Vg) are differentially regulated in wing and haltere discs. In wing discs, although Vg expression in non-DV cells is dependent on DV boundary function of Wg, it maintains its expression by autoregulation. Thus, overexpression of Vg in non-DV cells can bypass the requirement for Wg signaling from the DV boundary. Ubx functions, at least, at two levels to repress Vestigial expression in non-DV cells of haltere discs. At the DV boundary, it functions downstream of Shaggy/GSK3 beta to enhance the degradation of Armadillo (Arm), which causes downregulation of Wg signaling. In non-DV cells, Ubx inhibits event(s) downstream of Arm, but upstream of Vg autoregulation. Repression of Vg at multiple levels appears to be crucial for Ubx-mediated specification of the haltere fate. Overexpression of Vg in haltere discs is enough to override Ubx function and cause haltere-to-wing homeotic transformations.     

Drosophila glypicans Dally and Dally-like shape the extracellular Wingless morphogen gradient in the wing disc

Drosophila Wingless (Wg) is the founding member of the Wnt family of secreted proteins. During the wing development, Wg acts as a morphogen whose concentration gradient provides positional cues for wing patterning. The molecular mechanism(s) of Wg gradient formation is not fully understood. Here, we systematically analyzed the roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc. We demonstrate that both Dally and Dlp are essential and have different roles in Wg gradient formation. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. To our surprise, although Fz2 was suggested to play an essential role in Wg gradient formation by ectopic expression studies, removal of Fz2 activity does not alter the extracellular Wg gradient. Interestingly, removal of both Fz and Fz2, or Arr causes enhanced extracellular Wg levels, which is mainly resulted from upregulated Dlp levels. We further show that Notum, a negative regulator of Wg signaling, downregulates Wg signaling mainly by modifying Dally. Last, we demonstrate that Wg movement is impeded by cells mutant for both dally and dlp. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by combined actions of Dally and Dlp. We propose that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp in the wing disc.     

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal-ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO. Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. We have conducted a modular P-element based EP (enhancer/promoter) misexpression screen to look for genes involved in Apterous activity. We have found Osa, a member of the Brahma chromatin-remodeling complex, as a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. We propose that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression.     

Disruption of C-terminal cytoplasmic domain of betaPS integrin subunit has dominant negative properties in developing Drosophila

We have analyzed a set of new and existing strong mutations in the myospheroid gene, which encodes the betaPS integrin subunit of Drosophila. In addition to missense and other null mutations, three mutants behave as antimorphic alleles, indicative of dominant negative properties. Unlike null alleles, the three antimorphic mutants are synthetically lethal in double heterozygotes with an inflated (alphaPS2) null allele, and they fail to complement very weak, otherwise viable alleles of myospheroid. Two of the antimorphs result from identical splice site lesions, which create a frameshift in the C-terminal half of the cytoplasmic domain of betaPS. The third antimorphic mutation is caused by a stop codon just before the cytoplasmic splice site. These mutant betaPS proteins can support cell spreading in culture, especially under conditions that appear to promote integrin activation. Analyses of developing animals indicate that the dominant negative properties are not a result of inefficient surface expression, or simple competition between functional and nonfunctional proteins. These data indicate that mutations disrupting the C-terminal cytoplasmic domain of integrin beta subunits can have dominant negative effects in situ, at normal levels of expression, and that this property does not necessarily depend on a specific new protein sequence or structure. The results are discussed with respect to similar vertebrate beta subunit cytoplasmic mutations.     

Temporally dynamic response to Wingless directs the sequential elaboration of the proximodistal axis of the Drosophila wing

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. Here, we show that a temporally dynamic response to Wingless signalling sequentially elaborates the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate, zfh-2, is initially activated by Wingless throughout the "wing primordium," but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. Our data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance.     

Endocytic trafficking of Wingless and its receptors, Arrow and DFrizzled-2, in the Drosophila wing

During animal development, Wnt/Wingless (Wg) signaling is required for the patterning of multiple tissues. While insufficient signal transduction is detrimental to normal development, ectopic activation of the pathway can be just as devastating. Thus, numerous controls exist to precisely regulate Wg signaling levels. Endocytic trafficking of pathway components has recently been proposed as one such control mechanism. Here, we characterize the vesicular trafficking of Wg and its receptors, Arrow and DFrizzled-2 (DFz2), and investigate whether trafficking is important to regulate Wg signaling during dorsoventral patterning of the larval wing. We demonstrate a role for Arrow and DFz2 in Wg internalization. Subsequently, Wg, Arrow and DFz2 are trafficked through the endocytic pathway to the lysosome, where they are degraded in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner. Surprisingly, we find that Wg signaling is not attenuated by lysosomal targeting in the wing disc. Rather, we suggest that signaling is dampened intracellularly at an earlier trafficking step. This is in contrast to patterning of the embryonic epidermis, where lysosomal targeting is required to restrict the range of Wg signaling. Thus, signal modulation by endocytic routing will depend on the tissue to be patterned and the goals during that patterning event.     

Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development in Drosophila

Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin (DE-Cad) regulates epithelial cell-cell adhesion at adherens junctions (AJs). Here, we show that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless (Wg), a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of beta-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, we show that Ft intersects Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing.     

Changing spatial patterns of DNA replication in the developing wing of Drosophila

Using an antibody against bromodeoxyuridine we have analyzed the distribution of S-phase nuclei in the wing disc of Drosophila as the larval disc transforms into the adult wing during metamorphosis. On the basis of the timing of replication three cell populations can be distinguished: the cells of the presumptive wing margin, the precursor cells of the longitudinal veins, and those of the intervein regions. In each of these populations the cell cycle is first arrested and later resumes at a specific time, so that at each developmental time point a characteristic spatial pattern of S-phase nuclei is seen. An interpretation of these changing patterns in terms of vein formation, compartments, and neural development is offered.     

Neuron differentiation and axon growth in the developing wing of Drosophila melanogaster

Sensory neurons in the wing of Drosophila originate locally from epithelial cells and send their axons toward the base of the wing in two major bundles, the L1 and L3 nerves. We have estimated the birth times of a number of identified wing sensory neurons using an X-irradiation technique and have followed the appearance of their somata and axons by means of an immunohistochemical stain. These cells become immunoreactive and begin axon growth in a sequence which mirrors the sequence of their birth times. The earliest ones are born before pupariation and begin axonogenesis within 1 to 2 hr after the onset of metamorphosis; the last are born and differentiate some 12 to 14 hr later. The L1 and L3 nerves are formed in sections, with specific neurons pioneering defined stretches of the pathways during the period between 0 and 4 hr after pupariation (AP), and finally joining together around 12 hr AP. By 16 hr AP the adult complement of neurons is present and the adult peripheral nerve pattern has been established. Pathway establishment appears to be specified by multiple cues. In places where neurons differentiate in close proximity to one another, random filopodial exploration followed by axon growth to a neighboring neuron soma might be the major factor leading to pathway construction. In other locations, filopodial contact between neighboring somata does not appear to occur, and axon pathways joining neural neighbors by the most direct route are not established. We propose that in these cases additional factors, including veins which are already present at the time of axonogenesis, influence the growth of axons through non-neural tissues.     

The scabrous protein can act as an extracellular antagonist of notch signaling in the Drosophila wing

Notch (N) is a receptor for signals that inhibit neural precursor specification [1-6]. As N and its ligand Delta (DI) are expressed homogeneously, other molecules may be differentially expressed or active to permit neural precursor cells to arise intermingled with nonneural cells [7,8]. During Drosophila wing development, the glycosyltransferase encoded by the gene fringe (fng) promotes N signaling in response to DI, but inhibits N signaling in response to Serrate (Ser), which encodes a ligand that is structurally similar to DI. Dorsal expression of Fng protein localizes N signaling to the dorsoventral (DV) wing margin [9-11]. The secreted protein Scabrous (Sca) is a candidate for modulation of N in neural cells. Mutations at the scabrous (sca) locus alter the locations where precursor cells form in the peripheral nervous system [12,13]. Unlike fringe, sca mutations act cell non-autonomously [12]. Here, we report that targeted misexpression of Sca during wing development inhibited N signaling, blocking expression of all N target genes. Sca reduced N activation in response to DI more than in response to Ser. Ligand-independent signaling by overexpression of N protein, or by expression of activated truncated N molecules, was not inhibited by Sca. Our results indicate that Sca can act on N to reduce its availability for paracrine and autocrine interactions with DI and Ser, and can act as an antagonist of N signaling.